The goal of this grant is to investigate the melanoma-forming potential of melanocyte stem cells (McSCs) and their progeny, thereby understanding initial events of melanomagenesis. The incidence of melanoma is increasing in the United States, presumably due to increased skin exposure to UV radiation. Currently, there is no way to cure melanoma when it becomes metastatic. Developing targets for early intervention and effective strategies for early diagnosis and prevention are necessary, however the mechanism involved in melanoma initiation is elusive. In mouse melanoma models, we can induce oncogenic mutations in melanocytes. However, melanocytes in mice primarily reside within the hair follicle and their behavior alters during the hair cycle, making it difficult to distinguish te changes caused by oncogenic mutations. Currently, no study has addressed when and how these oncogenic mutations exert its effect on melanocytes in vivo. Despite the belief that stem cells may be the best candidate for the cell of origin in tumors, the involvement of McSCs in melanoma remains unknown. Since McSCs are located in the hair follicle and their primary function is to generate hair melanocytes whereas most melanomas arise from inter-follicular epidermis, this population has been under-studied in melanoma studies. We found that follicular McSCs give rise to epidermal melanocytes in adult mice following UVB-radiation. We hypothesize that McSCs in the bulge may be the origin of epidermal melanocytes that are responsible for melanoma. In Aim 1, we will use genetic mouse models to induce oncogenic mutations (BRAF activation and PTEN loss that are often found in human melanoma) in McSCs. We will also utilize a genetically tractable fluorescent reporter to trace the fate and behavior of the mutated melanocyte. Utilizing these mice, we will carefully delineate how mutations induce change in proliferation, differentiation and migration status of melanocytes in the bulge and epidermis at each stage of the hair follicle cycle. These analyses will be the first to capture the initial alterations in melanocytes driven by oncogenic mutations and to determine the incidence of melanomagenesis from distinct populations of melanocytes. In Aim 2, we will isolate the melanocytes from distinct locations to examine their potential to produce melanoma upon transplantation and induction of the mutations in immunodeficient mice. By analyzing the incidence of melanoma in each specific population, we will determine whether there are intrinsic differences that cause melanoma formation. Elucidating the cell of origin through these in situ and ex vivo studies will ultimately help identification of novel strategies to prevent melanomagenesis.